PROJECT SUMMARY/ABSTRACT The immune system relies on the intrinsic ability of immune cells to respond to activating or inhibitory signals. In the context of tissues, many of these signals are derived from neighboring non-immune cells sharing the same immunomodulatory environment. Elucidating the links between spatial and functional cellular interactions represents a major contemporary challenge, in particular to functional characterization of immunological systems. Whereas several mechanisms of tissue-immune cell crosstalk have been established, this area has not been systematically studied and the basic principles governing tissue immune regulation remain largely unknown. Sepsis is a life-threatening condition characterized by profound dysregulation of both the immune system and tissue homeostasis, ultimately converging on severe organ dysfunction. In particular, failure of endothelial and epithelial barriers is a hallmark of sepsis-associated organ dysfunction and death. Recent emerging paradigms describing sepsis pathogenesis highlight the interdependency of immune homeostasis and tissue homeostasis and suggest that better understanding of mechanisms of tissue immune regulation is critical for devising effective treatment strategies for sepsis. Despite the growing appreciation of the crosstalk between cells and organs affected by sepsis, how tissues regulate antibacterial immunity in sepsis is poorly understood. Our laboratory uses advanced mouse models and imaging platforms to study how tissues orchestrate the immune response in the setting of immunity and inflammation. Through this proposal, we will develop and validate novel concepts and platforms for interrogating 1) the liver sinusoid as an immunoregulatory unit in sepsis; 2) epithelial inflammatory cytokines as guardians of the intestinal barrier in sepsis; and 3) intestinal mesenchymal cells as critical modulators of local and systemic bacterial infection. Our studies will define the immunological crosstalk established between tissue non-immune and immune cells and unravel the complex multicellular mechanisms orchestrating tissue immune responses in bacterial sepsis. Ultimately, these studies are aimed to establish the paradigm of tissue immune regulation in sepsis and advance the conceptual and mechanistic understanding of the tissue-level immune pathways regulating antibacterial immunity.